The invention relates generally to method and apparatus for flow cytometry, and, more particularly, to signal processing and data acquisition for multichannel (including multibeam) flow cytometers.
Flow cytometry is a powerful tool for biological analysis. In a flow cytometer a stream of particles (e.g., cells or cell fragments) which have been chemically tagged, i.e., with a fluorescent dye, flows through an illumination (laser) beam which causes the chemical tag to fluoresce. The light pulses (scatter and fluorescence) provide an identifying signature of the particle. A multilaser flow cytometer uses a plurality of spaced beams each of a different wavelength to excite different fluorescent dyes. Thus more information can be obtained using a multilaser flow cytometer since each cell can be probed successively by each beam to provide information relating to a multitude of characteristics. However, data collection in multibeam systems is operationally more complex because of the time separation between the beams and the uncertain correlation between signals from each beam. Similarly, multiple detectors can be used with each beam, creating similar multichannel data collection problems.
If the signals are obtained with multiple excitation beams, the pulses from a single particle will reach different detectors at different times. The asynchronous events can be correlated either before or after the pulse digitization. One prior art approach to pre-processing synchronization is to hold the pulse values in analog circuits until all measurements of an event have been completed (FIG. 1A). After the event leaves the last measurement beam, the held values are input to AD converters. As shown, the height from the pulse from the first measurement (beam 1) is held until the particle has passed the second illumination point (beam 2). Both pulse heights are then converted into a digitized value, either by a single multiplexed ADC or by two converters working in parallel. The time of measurement cycle (cycle time) is the beam separation time plus the AD conversion time. This has the disadvantage that an event occupies the acquisition electronics for the time it takes to traverse all excitation beams. Thus, in a multilaser flow cytometer, parameter synchronization by sample hold circuits greatly reduces the maximum throughput rate of the system. It is more efficient to delay the earliest pulses with analog delay lines such that all signals enter the acquisition channels simultaneously (FIG. 1B). As shown, the signal from the first beam is delayed with an analog delay line so the signals from the two beams arrive simultaneously at the pulse processing electronics The cycle time is the AD conversion time plus the pulse width. However, analog delay lines have some drawbacks. They are expensive. They may induce signal distortion. They become unmanageable for large numbers of detectors or long delay times.
Flow cytometrists increasingly conceive of meaningful experiments that require multiple illumination beams with several detectors per light source. They demand high sort and analysis rates. At the same time, they expect instruments to accurately identify particles that occur at very low frequencies. A few examples of such experiments are: drug uptake by individual cell populations in complex cell mixtures, chromosome sorting and analysis, and the detection of aberrant cells in a large population of normal cells. Such applications require data acquisition systems with multiple input channels and precisely defined timing protocols. The electronics must be fast and accurate.